The present invention relates to methods of expansion of renewable stem cells, to expanded populations of renewable stem cells and to their uses. In particular, the present invention relates to methods of reducing the expression and/or activity of CD38. In one embodiment, ex-vivo and/or in-vivo stem cell expansion is achieved according to the present invention by downregulation of retinoic acid receptor (RAR), retinoid X receptor (RXR), and/or Vitamin D receptor (VDR) signaling, either at the protein level via RAR, RXR and/or VDR antagonists or at the expression level via genetic engineering techniques, such as small interfering RNA (siRNA) techniques. In another embodiment, ex-vivo and/or in-vivo stem cell expansion is achieved according to the present invention by downregulation of CD38 either at the protein level via CD38 inhibitors, such as, for example, nicotinamide, or at the expression level via genetic engineering techniques, such as small interfering RNA (siRNA) techniques. The present invention further relates to therapeutic applications in which these methods and/or the expanded stem cells populations obtained thereby are utilized.
An increasing need for ex-vivo cultures of hematopoietic and non-hematopoietic stem cells has arisen, in particular for purposes such as stem cell expansion and retroviral-mediated gene transduction. Methods for generating ex-vivo cultures of stem cells to date, however, result in a rapid decline in stem cell population activity, further resulting in a markedly impaired self renewal potential and diminished transplantability of the cultured cell populations. The need to improve such methods is obvious. Additionally, applications in gene therapy using retroviral vectors necessitate the use of proliferating hematopoietic stem cells, yet require that these cells remain undifferentiated while in culture, in order to maintain long-term expression of the transduced gene. Thus, the ability to maintain ex-vivo cultures of hematopoietic and non-hematopoietic stem cell populations with long-term, self-renewal capacity is of critical importance for a wide array of medical therapeutic applications.
Presently, expansion of renewable stem cells have been achieved either by growing the stem cells over a feeder layer of fibroblast cells, or by growing the cells in the presence of the early acting cytokines thrombopoietin (TPO), interleukin-6 (IL-6), an FLT-3 ligand and stem cell factor (SCF) (Madlambayan G J et al (2001) J Hematother Stem Cell Res 10: 481, Punzel M et al (1999) Leukemia 13: 92, and Lange W et al (1996) Leukemia 10: 943). While expanding stem cells over a feeder layer results in vast, substantially endless cell expansion, expanding stem cells without a feeder layer, in the presence of the early acting cytokines, results in an elevated degree of differentiation (see controls described in the Examples section and Leslie N R et al (Blood (1998) 92: 4798), Petzer A L et al (1996) J Exp Med Jun 183: 2551, Kawa Y et al (2000) Pigment Cell Res 8: 73).
In any case, using present day technology, stem cells cannot be expanded unless first substantially enriched or isolated to homogeneity.
The art presently fails to teach an efficient method for expansion of renewable stem cells without a feeder layer.
CD38 is a member of an emerging family of cytosolic and membrane-bound enzymes whose substrate is nicotinamide adenine dinucleotide (NAD), a coenzyme ubiquitously distributed in nature. In human, CD38 is a 45 kDa type H trans-membrane glycoprotein. Recently, it has been demonstrated that CD38 is a multifunctional enzyme that exerts both NAD+ glycohydrolase activity and ADP-ribosyl cyclase activity and is thus able to produce nicotinamide, ADP-ribose (ADPR), cyclic-ADPR (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) from its substrates (Howard et al., 1993 Science 252:1056-1059; Lee et al., 1999 Biol. Chem. 380;785-793). The soluble domain of human CD38 catalyzes the conversion of NAD+ to cyclic ADP-ribose and to ADP-ribose via a common covalent intermediate (Sauve, A. A., Deng, H. T., Angelletti, R. H., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 7855-7859).
However, it was further found that CD38 is not characterized only by multi enzymatic activity but is further able to mobilize calcium, to transduce signals and to adhere to hyaluronan and to other ligands. Interaction with CD38 on various leukocyte subpopulation has profound though diverse effects on their life-span (Funaro A, Malavasi F J Biol Regul Homeost Agents 1999 January-March;13(1):54-61 Human CD38, a surface receptor, an enzyme, an adhesion molecule and not a simple marker).
CD38 is widely expressed in both hematopoietic and non hematopoietically-derived cells. Homologues of CD38 have also been found to be expressed in mammalian stromal cells (Bst-1) and in cells isolated from the invertebrate Aplysia californica (Prasad G S, 1996, nature Structural Biol 3:957-964).
Two of the metabolites produced by CD38, cADPR and NAADP, have been shown to induce the release of intracellular calcium in cells isolated from tissues of plants, invertebrates and mammals, suggesting that these metabolites may be global regulators of calcium responses (Lee et al., 1999 Biol. Chem. 380;785-793). Both cADPR and NAADP are known to induce calcium release from calcium stores that are distinct from those controlled by Ip3 receptors (Clapper, D L et al., 1987, J. Biological Chem. 262:9561-9568).
Hence, CD38, being the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo.
Nucleoplasmic calcium ions (Ca+2) influence highly important nuclear functions such as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, which are types of Ca+2 channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca+2 is still unclear.
It was found that CD38/ADP-ribosyl cyclase has its catalytic site within the nucleoplasm and hence it catalyses the intranuclear cyclization of NAD+, to produce nucleoplasmic cADPR. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca+2 release (Adebanjo O A et al. Nat Cell Biol 1999 November;1(7):409-14 A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis).
It was further found that agonists of ryanodine receptors sensitize cADPR-mediated calcium release and antagonists of ryanodine receptors block cADPR-dependent calcium release (Galione A et al., 1991, Science 253:143-146). Thus, it has been proposed that cADPR is likely to regulate calcium responses in tissues such as muscle and pancreas, where ryanodine receptors are expressed (Day et al., 2000 Parasitol 120:417-422; Silva et al., 1998, Biochem. Pharmacol 56:997-1003). It has been also shown that in mammalian smooth muscle cells, the calcium release in response to acetylcholine can be blocked not only with ryanodine receptor antagonists, but also with specific antagonists of cADPR such as 8-NH2-cADPR or 8-Br-cADPR (Guse, A H, 1999, Cell. Signal. 11:309-316). These findings, as well as others, indicate that ryanodine receptor agonists/antagonists such as cADPR can regulate calcium responses in cells isolated from diverse species.
As is discussed hereinabove, self-renewal of hemopoietic stem and progenitor cells (HPC), both in vivo and in vitro, is limited by cell differentiation. Differentiation in the hematopoietic system involves, among other changes, altered expression of surface antigens (Sieff C, Bicknell D, Caine G, Robinson J, Lam G, Greaves M F (1982) Changes in cell surface antigen expression during hematopoietic differentiation. Blood 60:703). In normal human, most of the hematopoietic pluripotent stem cells and the lineage committed progenitor cells are CD34+. The majority of cells are CD34+CD38+, with a minority of cells (<10%) being CD34+CD38−. The CD34+CD38− phenotype appears to identify the most immature hematopoietic cells, which are capable of self-renewal and multilineage differentiation. The CD34+CD38− cell fraction contains more long-term culture initiating cells (LTC-IC) pre-CFU and exhibits longer maintenance of their phenotype and delayed proliferative response to cytokines as compared with CD34+CD38+cells. CD34+CD38− can give rise to lymphoid and myeloid cells in vitro and have an enhanced capacity to repopulate SCID mice (Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick J E (1997) Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci USA 94:5320). Moreover, in patients who received autologous blood cell transplantation, the number of CD34+CD38− cells infused correlated positively with the speed of hematopoietic recovery. In line with these functional features, CD34+CD38− cells have been shown to have detectable levels of telomerase.
Recently, it has been reported that granulocytic differentiation of human HL-60 cells (a committed cell line) can be induced by retinoic acid and is accompanied by a massive expression of CD38. Concomitant with CD38 expression was the accumulation of cADPR, and both time courses preceded the onset of differentiation, suggesting a causal role for CD38. Consistently, treatment of HL-60 cells with a permeant inhibitor of CD38, nicotinamide, inhibited both the CD38 activity and differentiation. More specific blockage of CD38 expression was achieved by using morpholino antisense oligonucleotides targeting its mRNA, which produced a corresponding inhibition of differentiation as well (Munshi C B, Graeff R, Lee H C, J Biol Chem 2002 Dec. 20;277(51):49453-8).
In view of the findings described above with respect to the effect of CD38 on cADPR and ryanodine signal transduction pathways and hence on cell expansion and differentiation, the present inventors have envisioned that by modulating the expression and/or the activity of CD38, the expansion and differentiation of stem cells could be controlled. In particular, it was hypothesized that by reducing the expression and/or the activity of CD38, using agents that downregulate the expression of CD38 or inhibit the activity thereof, expansion of renewable stem cells, devoid of differentiation, would be achievable.
Nicotinamide (NA) is a water-soluble derivative of vitamin B, whose physiological active forms are nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH). The physiological active forms of NA serve as coenzyme in a variety of important metabolic reactions. Nicotinamide is further known to inhibit the enzymatic activity of CD38, to thereby affect the cADPR signal transduction pathway, a feature which is demonstrated, for example, in the studies described hereinabove (see, for example, Munshi C B, Graeff R, Lee H C, J Biol Chem 2002 Dec. 20;277(51):49453-8).
Hence, while conceiving the present invention, it was hypothesized that nicotinamide, as well as other agents known to inhibit the enzymatic activity of CD38, can be utilized for expanding stem cell populations while inhibiting the differentiation of the stem cells. It was further hypothesized that other small molecules, which are capable of interfering, directly or indirectly, with the expression of CD38 can be similarly used.
Retinoic acid (RA), the natural acidic derivative of Vitamin A (retinol) is an important regulator of embryonic development and it also influences the growth and differentiation of a wide variety of adult cell types. The biological effects of RA are generally mediated through their interaction with specific ligand-activated nuclear transcription factors, their cognate RA receptors (RARs). Receptors of the retinoic acid family comprise RARS, RXRs, Vitamin D receptors (VDRs), thyroid hormone receptors (THRs) and others. When activated by specific ligands these receptors behave as transcription factors, controlling gene expression during embryonic and adult development. The RAR and RXR families of receptors uniquely exhibit modular structures harboring distinct DNA-binding and ligand-binding domains. These receptors probably mediate their biological effects by binding to regulatory elements (e.g., retinoic acid response elements, or RAREs) as RAR-RXR heterodimers that are present in the promoters of their specific target genes (1, 2, 3).
Retinoid receptors thus behave as ligand-dependent transcriptional regulators, repressing transcription in the absence of ligand and activating transcription in its presence. These divergent effects on transcription are mediated through the recruitment of co-regulators: un-liganded receptors bind corepressors (NCOR and SMRT) that are found within a complex exhibiting histone deacetylase (HDAC) activity, whereas liganded receptors recruit co-activators with histone acetylase activity (HATs). Chromatin remodeling may also be required, suggesting a hierarchy of promoter structure modifications in RA target genes carried out by multiple co-regulatory complexes.
The first retinoic acid receptor identified, designated RAR-alpha, modulates transcription of specific target genes in a manner which is ligand-dependent, as subsequently shown for many of the members of the steroid/thyroid hormone intracellular receptor superfamily. The endogenous low-molecular-weight ligand, upon which the transcription-modulating activity of RAR-alpha depends, is all-trans-retinoic acid. Retinoic acid receptor-mediated changes in gene expression result in characteristic alterations in cellular phenotype, affecting multiple tissues. Additional RAR-alpha related genes have been identified, designated RAR-beta and RAR-gamma, and exhibit a high level of homology to RAR-alpha and each other (4, 5). The ligand-binding region of the three RAR subtype receptors has a primary amino acid sequence divergence of less than 15%.
Similarly, additional members of the steroid/thyroid receptor superfamily responsive to retinoic acid have been identified (6), and have been designated as the retinoid X receptor (RXR) family. Like the RARs, the RXRs are also known to comprise at least three subtypes or isoforms, namely RXR-alpha, RXR-beta, and RXR-gamma, with corresponding unique patterns of expression (7).
Although both the RARs and RXRs bind the ligand all-trans-retinoic acid in vivo, the receptors differ in several important aspects. First, the RARs and RXRs significantly differ in their primary structure, especially regarding their ligand binding domains (e.g., alpha domains exhibit a mere 27% shared amino acid identity). These structural differences manifest in their differing relative degrees of responsiveness to various Vitamin A metabolites and synthetic retinoids. Additionally, tissue distribution patterns are distinctly different for RARs and RXRs. RARs and RXRs exhibit different target gene specificity. One example is regarding the cellular retinal binding protein type II (CRBPII) and apolipoprotein AI proteins that confer responsiveness to RXR, but not RAR. Furthermore, RAR has also been shown to repress RXR-mediated activation through the CRBPII RXR response element (8). These data indicate that the two separate retinoic acid responsive pathways are not simply redundant, but instead manifest a complex interplay.
Vitamin D (VitD) is an additional potent activator of one of the receptors belonging to the retinoid receptor superfamily. The nuclear hormone 1 alpha, 25-dihydroxyvitamin D (3) (1 alpha, 25 (OH) (2) D (3)) binds its cognate receptor (VDR) and acts as a transcription factor when in combined contact with the retinoid X receptor (RXR), coactivator proteins, and specific DNA binding sites (VDREs). Ligand-mediated conformational changes of the VDR comprise the molecular switch controlling nuclear 1 alpha, 25 (OH) (2) D (3), signaling events.
Cell-specific VDR antagonists reveal the exquisite control and regulation of the pleiotropic 1 alpha, 25 (OH) (2) D (3) endocrine system, with consequences in maintenance of calcium homeostasis, bone mineralization and other cellular functions. Antagonists to VitD were shown to act via the same mechanism: they selectively stabilize an antagonistic conformation of the ligand-binding domain of the VDR within VDR-RXR-VDRE complexes, inhibiting the interaction of the VDR with coactivator proteins and induction of transactivation. Interestingly, cells treated with VitD antagonists contain VDR-RXR heterodimers in different conformations as compared to cells stimulated with VitD agonists (16).
Retinoic acid and VitD can cooperatively stimulate transcriptional events involving a common DNA binding site or hormone response element (HRE). Conversely, VDR/RXR heterodimers have been found to bind without defined polarity and in a transcriptionally unproductive manner to certain RA response elements, and under these circumstances Vitamin D inhibits the response to RA. Although competition for binding to DNA may contribute to this inhibitory response, titration of common coactivators by VDR also appears to be involved in this trans-repression. Therefore, the regulation of the transcriptional response to RA and VitD is dependent upon a complex combinatory pattern of interaction among the different receptors, co-activators (17) and their binding to the appropriate DNA binding sites.
In parallel to their function as transcriptional regulators, retinoid receptors such as RAR and RXR play important roles in regulating the growth and differentiation of a variety of cell-types, as well (18). RAR agonists such as all-trans-retinoic acid (ATRA) are predominantly known for their effects in inducing cell-differentiation, as seen in experiments utilizing malignant cancer cells and embryonic stem cells (19), where potent induction of terminal differentiation was evident. Cell differentiation is not an exclusive result, however, as RA has been shown to exhibit different effects on cultured hematopoietic cells, depending on their maturational state (20). While retinoids accelerated the growth and differentiation of granulocyte progenitors in cytokine-stimulated cultures of purified CD34+ cells, use of stem cells produced an opposite effect (42). Retinoid treatment has also been shown to inhibit differentiation of pre-adipose cells (43).
Whereas the RAR antagonist AGN 193109 exerted a positive effect on the differentiation of hematopoietic stem cells (41) the RAR agonist 4-[4-(4-ethylphenyl)dimethyl-chromen-yl]ethynyl}-benzoic acid] functions in an opposing manner. Conversely, RAR antagonists have been shown to prevent granulocytic differentiation in experiments utilizing the promyelocytic cell line, HL-60 (41). Similarly, creation of myeloid cell lines defective in signaling through their retinoid receptors do not undergo granulocytic differentiation in the presence of G-CSF (22), and retinoid-deficient tissues acquire a pre-malignant phenotype, and a concomitant loss of differentiation (29, 30). Malignant cell lines derived from various carcinomas exhibit diminished expression of retinoic acid receptor mRNA, implying that the loss of expression may be an important event in tumorogenesis (33, 34, 35, 36, 37). Furthermore, disruption of retinoic acid receptor activity, as evidenced in knock-out mouse models disrupted for the RAR gene, display an in vitro block to granulocytic differentiation (38, 39).
However, other studies using a similar approach have resulted in the development of hematopoietic cell lines (23). The hematopoietic stem and early progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34+, and exclusion of expression of the surface lineage antigen markers, Lin−. Experiments utilizing several leukemia cell lines revealed that retinoic acid receptor mediated signaling results in the induction of expression of the differentiation marker CD38 cell surface antigen whereas antagonists to RAR abolished CD38 antigen up-regulation (24, 25).
Therefore, to date, the data are conflicting as to definitive roles for VitD and RA in induction of myelomonocytic and promyelocytic cell differentiation, or prevention of these processes. Although some previous studies with inactivation of RAR, RXR and VDR using antagonists, antisense technology or transduction methods with truncated receptors, yielded inhibited granulocytic and monocytic differentiation, these studies were conducted using leukemia cell lines that are blocked at the myeloblast or promyelocytic stage of differentiation (19, 22, 64). As stated above, isolation procedures for hematopoietic and other stem cells result in small populations of cells that are difficult to expand in ex-vivo cultures. Current culture methods enable large-scale expansion of progenitor and differentiated cell populations, but provide minimal amplification of the stem cell component. Applications and uses of stem cell populations for cell replacement therapy, in-vivo tissue regeneration, ex-vivo tissue formation and gene therapy, necessitate the acquirement of large numbers of these cell populations.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of propagating large numbers of stem cells in an ex-vivo setting. Methods enabling ex-vivo expansion of stem cell compartments yielding large numbers of these cell populations will therefore pioneer feasible stem cell therapies for human treatment, with a clear and direct impact on the treatment of an infinite number of pathologies and diseases.
Some pathological and medically induced conditions are characterized by a low number of in-vivo self or transplanted renewable stem cells, in which conditions, it will be advantageous to have an agent which can induce stem cell expansion in-vivo.